Synthesis and biological evaluation of indomethacin analogs possessing a N-difluoromethyl-1,2-dihydropyrid-2-one ring system: A search for novel cyclooxygenase and lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.07.132

A novel class of indomethacin analogs were synthesized wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety (5-LOX pharmacophore) was attached at its C-4 or C-5 position via either a CO (14a-b) or CH₂ (19a-b) linker to the indole N¹-position. In this regard, replacement of the 4-chlorobenzoyl group present in indomethacin by N-difluoromethyl-1,2- dihydropyrid-2-one-4-(or 5-)carbonyl and N-difluoromethyl-1,2-dihydropyrid-2- one-4-yl(or 5-yl)methylene moieties furnished compounds showing no inhibitory activities against the COX-2/5-LOX enzymes (except for the weak but selective COX-2 inhibitor 19a, COX-2 IC₅₀ = 31 μM), and moderate in vivo anti-inflammatory activities (except for the methylene compound 19a that was inactive). These structure-activity data indicate replacement of the 4-chlorobenzoyl group present in indomethacin by a N-difluoromethyl-1,2- dihydropyrid-2-one ring system connected by a CO or CH₂ linker is not a suitable approach for the design of dual COX-2/5-LOX inhibitory analogs of indomethacin.

Full text of DOI:10.1016/j.bmcl.2010.07.132

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5776–5780
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of indomethacin analogs possessing a
N-difluoromethyl-1,2-dihydropyrid-2-one ring system: A search for novel
cyclooxygenase and lipoxygenase inhibitors
Morshed A. Chowdhury, Zhangjian Huang, Khaled R. A. Abdellatif, Ying Dong, Gang Yu, Carlos A. Velázquez,
*
Edward E. Knaus
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta, Canada T6G 2N8
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of indomethacin analogs were synthesized wherein a N-difluoromethyl-1,2-dihydropyrid-
2-one moiety (5-LOX pharmacophore) was attached at its C-4 or C-5 position via either a C@O (14a–b)
or CH2 (19a–b) linker to the indole N1-position. In this regard, replacement of the 4-chlorobenzoyl group
present in indomethacin by N-difluoromethyl-1,2-dihydropyrid-2-one-4-(or 5-)carbonyl and N-difluoro-
methyl-1,2-dihydropyrid-2-one-4-yl(or 5-yl)methylene moieties furnished compounds showing no
inhibitory activities against the COX-2/5-LOX enzymes (except for the weak but selective COX-2 inhibitor
Received 16 July 2010
Revised 29 July 2010
Accepted 30 July 2010
Available online 3 August 2010
Keywords:
Indomethacin analogs
N-Difluoromethyl-1,2-dihydropyrid-2-one
5-Lipoxygenase pharmacophore
Cyclooxygenase and 5-lipoxygenase
inhibition
19a, COX-2 IC50 = 31 lM), and moderate in vivo anti-inflammatory activities (except for the methylene
compound 19a that was inactive). These structure–activity data indicate replacement of the 4-chloroben-
zoyl group present in indomethacin by a N-difluoromethyl-1,2-dihydropyrid-2-one ring system con-
nected by a C@O or CH2 linker is not a suitable approach for the design of dual COX-2/5-LOX
inhibitory analogs of indomethacin.
Anti-inflammatory activity
Ó 2010 Elsevier Ltd. All rights reserved.
Arachidonic acid (AA), following its release from membrane-
bound phospholipids, undergoes biotransformation via the cyclo-
oxygenase (COX) and lipoxygenase (LOX) pathways. Proinflamma-
tory prostaglandins (PGs) produced via the COX pathway, and
leukotrienes (LTs) produced via the LOX pathway, are implicated
in physiological processes such as inflammation, fever, arthritis
and bronchospasm.1,2 PGs that cause contraindicated inflamma-
tion, fever, and pain are formed via the inducible COX-2 isozyme
whereas, PGs that regulate beneficial gastrointestinal cytoprotec-
tion and renal effects are produced via the constitutive COX-1 iso-
zyme.1–3 Alternatively, 5-LOX is associated with the production of
LTs that cause inflammatory, bronchoconstrictor, hypersensitivity,
anaphylactic and asthmatic actions. On the other hand, 15-LOX is
implicated in atherosclerosis because it catalyzes the oxidation of
lipoproteins (LDL, HDL) to atherogenic forms.4,5
acid (AA) cascade.1,9 It has been pointed out that inhibition of only
one of the COX/LOX pathways could shift the metabolism of AA to-
wards the other pathway, thereby inducing potential side effects.10
One of the more successful strategies to develop 5-LOX inhibitors
utilized hydroxamic acids and related N-hydroxyureas that act by
chelation of iron present in the 5-LOX enzyme.11 Two representa-
tive examples of iron chelating 5-LOX inhibitors include zileuton
(1)12 and tepoxalin (2)11 (see structures in Fig. 1). A potent hybrid
COX-2/5-LOX inhibitor (3) in which the C-3 trifluoromethyl substi-
tuent present in the COX-2 inhibitor celecoxib (4)13 was replaced
by a non-redox competitive 4-(3-fluoro-5-oxymethyl)phenyl-4-
methoxytetrahydropyran 5-LOX pharmacophore was reported by
Henichart and co-workers2 In recent studies, we described novel
classes of dual COX/5-LOX inhibitors that include celecoxib analogs
(5)14 and 1,2-diarylacetylene regioisomers (6).15 These compounds
5 and 6, that possess a N-difluoromethyl-1,2-dihydropyrid-2-one
5-LOX pharmacophore, exhibited effective AI activity. It was subse-
quently discovered that salicylic acid derivatives 7 possessing a N-
difluoromethyl-1,2-dihydropyrid-2-one moiety also exhibited dual
COX-2/5-LOX inhibitory activities.16 Accordingly, it was antici-
pated that replacement of the 4-chlorobenzoyl moiety in indo-
methacin (8) by N-difluoromethyl-1,2-dihydropyrid-2-one-4-(or
5-)carbonyl, or N-difluoromethyl-1,2-dihydropyrid-2-one-4-(or 5-
)ylmethylene, regioisomeric moieties may provide a hitherto un-
known class of dual COX-2/5-LOX inhibitory AI agents. As part of
this ongoing research program, we now describe the synthesis of
Dual inhibitors of the LOX/COX enzymatic pathways6 constitute
a rational approach for the design of more effective anti-inflamma-
tory agents with a superior safety profile relative to ulcerogenic
nonsteroidal anti-inflammatory drugs (NSAIDs), and selective
COX-2 inhibitors that increase the incidence of adverse cardiovas-
cular thrombotic effects.7,8 This view is based on a potentially
greater anti-inflammatory (AI) efficacy because of their ability to
synergistically block both metabolic pathways of the arachidonic
* Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492 1217.
E-mail address: eknaus@pharmacy.ualberta.ca (E.E. Knaus).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.132

M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5776–5780
5777
Me
N
MeO
MeO
MeO
HO
NH2 MeO
O
N
CH2CO2H
CH2CO2Bn
N
N
OH
a
b
O
S
Me
Me
N
H
10
N
H
9
Zileuton (1)
Tepoxalin (2)
Cl
MeO
CF3
CH2CO2Bn
CH2CO2Bn
O
MeO
N
N
N
Me
c
Me
d
N
C
N
C
N
O
Me
O
N
O
F
5
5
4
N
CHF2
4
SO2NH2
3
SO2Me
Cl
O
Celecoxib (4)
12a; 4-regioisomer
12b; 5-regioisomer
13a; 4-regioisomer
13b; 5-regioisomer
CF3
CHF2
N
MeO
R1O2S
CH2CO2H
N
N
N
C
C
O
F2HC
Me
O
N
C
6, R1 = Me, NH2
O
SO2R1
5, R1 = Me, NH2
5
4
N
CHF2
CH2CO2H
Me
MeO
O
O
N
14a; 4-regioisomer
14b; 5-regioisomer
R1
4
5
N
F2HC
Scheme 1. Reagents and conditions: (a) benzyl chloroformate, DMAP, Et3N, CH2Cl2,
0 °C, 0.5 h; (b) 2-chloropyridine-4-carbonyl chloride (11a) or 2-chloropyridine-5-
carbonyl chloride (11b), DMAP, Et3N, CH2Cl2, 25 °C, 60 h; (c) FSO2CF2COOH,
NaHCO3, MeCN, reflux, 40 h; (d) 10% Pd/C, EtOAc, MeOH, H2 (45 psi), 25 °C, 24 h.
CO2H
O
Cl
7, R1 = OH, SO2NHAc
Indomethacin (8)
Figure 1. Chemical structures of some representative iron-chelating 5-LOX inhib-
itors (1–2), a COX-2/5-LOX inhibitor (3), a selective COX-2 inhibitor (4), dual COX/5-
LOX inhibitors (5–7) and the non-selective COX-1/COX-2 inhibitor (8).
tion with acetyl chloride and methanol21 that afforded the methyl
ester (15)22 in 95% yield (Scheme 2). N1-alkylation of 15 with a 2-
chloro-4-(or 5-)chloromethylpyridine 16a or 16b in THF using
potassium t-butoxide as base19 furnished the respective coupled
product 17a or 17b in 34–49% yields as illustrated in Scheme 2.
Subsequent transformation of the 2-chloropyridine ring in 17a or
17b to a N-difluoromethyl-1,2-dihydropyrid-2-one ring present
in 18a or 18b was carried out in 34–53% yield using the same pro-
cedure described previously for the preparation of 13a–b. Depro-
tection of the CO2Me group in 18a and 18b with aqueous 2 N
lithium hydroxide in methanol-tetrahydrofuran23 furnished the
target [1-(N-difluoromethyl-1,2-dihydropyrid-2-one-4-(or 5-)yl-
methyl-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid regioisom-
ers (19a and 19b) in 59–85% yields.
The rational for the design of the [1-(N-difluoromethyl-1,2-
dihydropyrid-2-one-4-(or 5-)carbonyl)-5-methoxy-2-methyl-1H-
indol-3-yl]acetic acids (14a–b) and [1-(N-difluoromethyl-1,2-
dihydropyrid-2-one-4-(or 5-)ylmethylene-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acids (19a–b) was based on the expectation
that replacement of the 4-chlorobenzoyl moiety in indomethacin
by a N-difluoromethyl-1,2-dihydropyrid-2-one ring system in con-
junction with a carbonyl or methylene linker may provide a hith-
erto unknown class of compounds with dual COX-2/5-LOX
inhibitory activities. The CONCHF2 fragment of the N-difluoro-
methyl-1,2-dihydropyrid-2-one ring present in 14a–b and 19a–b
can be viewed as a cyclic hydroxamic acid mimetic. These N-difluo-
romethyl-1,2-dihydropyrid-2-ones 14a–b and 19a–b could inhibit
the 5-LOX enzyme by two possible mechanisms. In this regard
14a–b and 19a–b, like acyclic hydroxamic acids, may act as effec-
tive iron chelators to exhibit 5-LOX inhibitory activity. It has been
reported that there is a substantial build-up of negative potential
a novel class of indomethacin analogs wherein a N-difluoromethyl-
1,2-dihydropyrid-2-one moiety (5-LOX pharmacophore) was at-
tached at its C-4 or C-5 position via either a C@O (14a–b) or CH2
(19a–b) linker to the indole N1-position, their in vitro evaluation
as COX-1, COX-2, and 5-LOX inhibitors, and in vivo assessment
as AI agents.
A group of compounds where the 4-chlorobenzoyl group present
in indomethacin was replaced by a N-difluoromethyl-1,2-dihydro-
pyrid-2-one ring system attached via a carbonyl, or methylene, link-
ing group was synthesized using the reaction sequence illustrated in
Schemes 1 and 2, respectively. The carboxylic group of 5-methoxy-
2-methyl-3-indoleacetic acid (9) was protected using a procedure
analogous to a literature method17 that furnished the benzyl ester
(10)18 in 97% yield (Scheme 1). Reaction of 10 with 2-chloropyri-
dine-4-carbonyl chloride (11a), or its 5-regioisomer 11b, in the pres-
ence of 4-dimethylaminopyridine (DMAP) in triethylamine and
dichloromethane19 afforded the respective coupled product 12a or
12b in 95–97% yield. Subsequent transformation of the 2-chloropyr-
idine ring in compounds 12a and 12b upon treatment with 2,2-di-
fluoro-2-(fluorosulfonyl)acetic acid (FSO2CF2COOH)20 afforded the
respective N-difluoromethyl-1,2-dihydropyrid-2-one regioisomer
13a or 13b in 31–32% yields. Deprotection of the CO2Bn group in
13a and 13b with H2 (45 psi) in the presence of 10% Pd on carbon
in methanol–ethyl acetate19 afforded the target [1-(N-difluoro-
methyl-1,2-dihydropyrid-2-one-4-(and 5-)carbonyl)-5-methoxy-
2-methyl-1H-indol-3-yl]acetic acid regioisomers (14a and 14b) in
46–75% yields.
For the synthesis of the target compounds 19a–b having a
methylene linker, the carboxyl group in 9 was protected by reac-

5778
M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5776–5780
Table 1
MeO
CH2CO2Me
In vitro COX-1, COX-2, 5-LOX enzyme inhibition, and in vivo anti-inflammatory
activity, data for indomethacin analogs containing a N-difluoromethyl-1,2-dihydro-
pyrid-2-one-4- or 5-carbonyl moiety (14a–b) or N-difluoromethyl-1,2-dihydropyrid-
2-one-4- or 5-ylmethyl moiety (19a–b)
a
b
9
Me
N
H
MeO
MeO
15
CH2CO2H
CH2CO2H
MeO
MeO
CH2CO2Me
CH2CO2Me
Me
O
Me
N
C
N
CH2
Me
c
Me
d
N
N
5
5
CH2
CH2
4
N
CHF2
4
N
CHF2
5
5
4
N
4
N
CHF2
O
O
14a; 4-regioisomer
14b; 5-regioisomer
19a; 4-regioisomer
19b; 5-regioisomer
Cl
O
17a; 4-regioisomer
17b; 5-regioisomer
18a; 4-regioisomer
18b; 5-regioisomer
Compound
COX-1
IC50
COX-2
IC50
5-LOX
IC50
AI activity
% Inhibition
(
l
M)a
(
l
M)a
(
l
M)b
MeO
14a
14b
19a
19b
>100
>100
>100
>100
0.1
>100
>100
31
>100
5.7d
—
>100
>100
>100
>100
—
25.5 7c
18.9 6.4c
Inactivec
16.0 0.5c
50e
CH2CO2H
Me
N
Indomethacin
NDGAf
—
13
—
CH2
5
a
The in vitro test compound concentration required to produce 50% inhibition of
ovine COX-1 or human recombinant COX-2. The result (IC50 M) is the mean of two
4
N
CHF2
,
l
determinations acquired using the enzyme immunoassay kit (catalog no. 560131,
Cayman Chemicals Inc., Ann Arbor, MI), and the deviation from the mean is <10% of
the mean value.
The in vitro test compound concentration required to produce 50% inhibition of
potato 5-LOX (Cayman Chemicals Inc. catalog no. 60401). The result (IC50, lM) is
O
19a; 4-regioisomer
19b; 5-regioisomer
b
the mean of two determinations acquired using a LOX assay kit (catalog no. 760700,
Cayman Chemicals Inc., Ann Arbor, MI), and the deviation from the mean is <10% of
the mean value.
Scheme 2. Reagents and conditions: (a) acetyl chloride, MeOH, 0 °C and then 60 °C,
16 h; (b) 2-chloro-4-chloromethylpyridine (16a) or 2-chloro-5-chloromethylpyri-
dine (16b), t-BuOK, THF, reflux under argon, 60 h; (c) FSO2CF2COOH, NaHCO3,
MeCN, reflux, 40 h; (d) LiOH, THF–MeOH–H2O, reflux, 3 h.
c
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results
are expressed as the % inhibition of inflammation at 3 h following a 100 mg/kg oral
dose of the test compound.
d
Data acquired using ovine COX-2 (catalog no. 56101, Cayman Chemicals Inc.).
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results
e
around the two fluorine atoms of a CHF2 group.24 Despite this high
electron-density, an aliphatic fluorine seldom acts as a hydrogen-
bond acceptor, presumably due to its high electronegativity and
low polarizability.25,26 Therefore, it is also plausible that the CHF2
group may interact with a positively charged region on the enzyme
that may contribute to enhanced affinity and competitive revers-
ible inhibition of the COX and/or 5-LOX enzymes.27 In addition,
these cyclic N-difluoromethyl-1,2-dihydropyrid-2-ones, unlike
acyclic hydroxamic acids which undergo facile biotransformation
to the acids, are expected to have a greater metabolic stability with
increased oral efficacy. Although there is some distortion from pla-
narity at the N1-nitrogen atom of the N-difluoromethyl-1,2-dihyd-
ropyrid-2-one ring system, the relatively flat diene portion of this
quasi-planar ring system has the potential to serve as a suitable
replacement for the 4-chlorobenzoyl moiety present in indometh-
acin 8 resulting in retention of COX-2 inhibitory activity.
are expressed as the % inhibition of inflammation at 3 h following a 4.2 mg/kg oral
dose of the reference drug indomethacin.
f
NDGA, nordihydroguaiaretic acid.
observation that compounds 14a–b and 19b did not inhibit either
the COX-1 or COX-2 isozyme suggests that their weak AI effect may
occur by a non-COX mechanism to reduce inflammation.
In conclusion, a hitherto unknown class of [1-(N-difluoro-
methyl-1,2-dihydropyrid-2-one-4 or 5-carbonyl)-5-methoxy-2-
methyl-1H-indol-3-yl]acetic acids (14a–b) and [1-(N-difluoro-
methyl-1,2-dihydropyrid-2-one-4- or 5-ylmethyl)-5-methoxy-2-
methyl-1H-indol-3-yl]acetic acids (19a–b), was synthesized28 for
evaluation as dual 5-LOX29 and COX-1/COX-230 isozyme inhibitors
of inflammation.31 The structure–activity data acquired indicate
that coupling of a N-difluoromethyl-1,2-dihydropyrid-2-one ring
system via a carbonyl (C@O), or methylene (CH2), linker to the
N1-position of 5-methoxy-2-methyl-1H-indol-3-yl]acetic acids is
not a suitable strategy for the design of indomethacin analogs as
AI agents that act by inhibition of the 5-LOX and/or COX-2 en-
zymes. The observation that 14a–b and 19a–b are not effective
inhibitors of the COX and LOX enzymes indicates how structural
changes can unexpectedly abolish activity since previous studies
showed that celecoxib (5), linear acetylene (6) and salicylic acid
In vitro COX-1 and COX-2 enzyme inhibition studies (see data in
Table 1) showed that three indomethacin analogs (14a–b, 19b) did
not inhibit either the COX-1 or COX-2 isozyme at a 100
compound concentration. Although the C-4 regioisomer 19a did
not inhibit COX-1 (IC50 >100 M), it is a weak inhibitor of the
COX-2 isozyme (COX-2 IC50 = 31 M).
lM test
l
l
In vitro 5-LOX inhibition studies showed that all four indometh-
(7) analogs having
a N-difluoromethyl-1,2-dihydropyrid-2-one
acin analogs (14a–b, 19a–b) failed to inhibit the 5-LOX enzyme
moiety exhibited effective in vitro dual COX/LOX inhibitory, and
in vivo anti-inflammatory, activities.
(IC50 >100
acid (NDGA) (IC50 = 13
l
M) relative to the reference drug nordihydroguaiaretic
lM).
The AI activities exhibited by the indomethacin analogs 14a–b
and 19a–b were determined using a carrageenan-induced rat foot
paw edema model at a 100 mg/kg oral dose. In this assay, com-
pounds 14a–b, 19b showed moderate AI activity (16.0–25.5% inhi-
bition). In contrast, compound 19a showed no AI activity. The
Acknowledgment
We are grateful to the Canadian Institutes of Health Research
(Grant No. MOP-14712) for financial support of this research.

M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5776–5780
5779
7.15 (d, J = 8.5 Hz, 1H, H-7), 7.27–7.40 (m, 5H, C6H5), 7.80 (br s, 1H, NH,
exchangeable with D2O).
References and notes
General procedure for the synthesis of [1-(2-chloropyridine-4- or 5-carbonyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetic acid benzyl esters (12a–b): A solution of
the benzyl ester 10 (0.5 g, 1.62 mmol), 2-chloropyridine-4-(or 5-)carbonyl
chloride 11a or 11b (0.34 g, 1.94 mmol), DMAP (104 mg, 0.85 mmol), and
triethylamine (1.20 mL, 8.61 mmol) in dichloromethane (10 mL) was stirred at
25 °C for 60 h. Removal of solvents from the reaction mixture in vacuo gave
crude product that was purified by silica gel column chromatography using
hexanes–ethyl acetate (1:1, v/v) as eluent to furnish the respective title
compound 12a or 12b. Some physical and spectroscopic data for 12a–b are
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23. Adamski-Werner, S. L.; Palaninathan, S. K.; Sacchettini, J. C.; Kelly, J. W. J. Med.
Chem. 2004, 47, 355.
24. Narjes, F.; Koehler, K. F.; Koch, U.; Gerlach, B.; Colarusso, S.; Steinkühler, C.;
Brunetti, M.; Altamura, S.; De Francesco, R.; Matassa, V. G. Bioorg. Med. Chem.
Lett. 2002, 12, 701. and references cited therein.
25. Dunitz, J. D.; Taylor, R. Chem. A Eur. J. 1997, 3, 89.
26. Howard, J. A. K.; Hoy, V. J.; O’Hagan, D.; Smith, G. T. Tetrahedron 1996, 52,
12613.
benzyl ester (12a): Product 12a was obtained as a yellow oil in 95% yield; IR
(film) 1735 (ester), 1685 (amide) cmÀ1 1H NMR (CDCl3) d 2.33 (s, 3H, CH3),
;
3.71 (s, 2H, CH2CO2Bn), 3.77 (s, 3H, OCH3), 5.15 (s, 2H, CH2Ph), 6.72 (dd, J = 2.4,
9.1 Hz, 1H, indole H-6), 6.93 (d, J = 2.4 Hz, 1H, indole H-4), 7.01 (d, J = 9.1 Hz,
1H, indole H-7), 7.27–7.39 (m, 5H, C6H5), 7.43 (dd, J = 1.2, 4.9 Hz, 1H, pyridyl H-
5), 7.59 (d, J = 1.2 Hz, 1H, pyridyl H-3), 8.58 (d, J = 4.9 Hz, 1H, pyridyl H-6); 13
C
NMR (CDCl3) d 13.9, 30.4, 55.6, 66.9, 101.7, 112.3, 114.1, 115.2, 121.2, 123.5,
128.2, 128.4, 128.6, 130.2, 131.0, 135.3, 135.6, 146.0, 150.6, 152.6, 156.6, 166.0,
170.3.
[1-(2-Chloropyridine-5-carbonyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
benzyl ester (12b): Product 12b was obtained as a yellow oil in 97% yield; IR
(film) 1735 (ester), 1683 (amide) cmÀ1 1H NMR (CDCl3) d 2.37 (s, 3H, CH3),
;
3.72 (s, 2H, CH2CO2Bn), 3.77 (s, 3H, OCH3), 5.15 (s, 2H, CH2Ph), 6.71 (dd, J = 2.4,
9.1 Hz, 1H, indole H-6), 6.93 (d, J = 2.4 Hz, 1H, indole H-4), 6.94 (d, J = 9.1 Hz,
1H, indole H-7), 7.27–7.39 (m, 5H, C6H5), 7.47 (d, J = 8.5 Hz, 1H, pyridyl H-3),
7.96 (dd, J = 2.4, 8.5 Hz, pyridyl H-4), 8.70 ((d, J = 2.4 Hz, 1H, pyridyl H-6); 13C
NMR (CDCl3) d 13.7, 30.4, 55.6, 66.9, 101.6, 112.1, 113.4, 114.9, 124.4, 128.2,
128.4, 128.5, 130.3, 130.5, 130.8, 135.5, 135.6, 139.5, 150.7, 155.3, 156.4, 166.0,
170.4.
General procedure for the synthesis of [1-(N-difluoromethyl-1,2-dihydropyrid-2-
one-4- or 5-carbonyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid benzyl
esters (13a–b): FSO2CF2CO2H (0.72 g, 4.02 mmol), and then NaHCO3 (113 mg,
1.34 mmol), was added to a solution of a 2-chloropyridine 12a or 12b (0.60 g,
1.34 mmol) in MeCN (10 mL). This mixture was then heated at reflux under
argon for 40 h, cooled to 25 °C, a saturated solution of aqueous NaHCO3
(10 mL) was added, and the mixture was extracted with EtOAc (3 Â 20 mL).
The combined organic extracts were successively washed with water (25 mL)
and brine, and the organic fraction was dried (MgSO4). Filtration and then
removal of the solvent from the organic fraction in vacuo afforded the impure
product which was purified by silica gel chromatography using hexanes–ethyl
acetate (2:1, v/v) as eluent to furnish the title compound 13a or 13b. Some
physical and spectroscopic data for 13a–b are listed below.
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-4-carbonyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid benzyl ester (13a): Product 13a was obtained as a
yellow oil in 32% yield; IR (film) 1741 (ester), 1689 (amide) cmÀ1 1H NMR
;
(CDCl3) d 2.41 (s, 3H, CH3), 3.69 (s, 2H, CH2CO2Bn), 3.78 (s, 3H, OCH3), 5.14 (s,
2H, CH2Ph), 6.46 (dd, J = 1.8, 7.3 Hz, 1H, pyridone H-5), 6.78 (d, J = 1.8 Hz, 1H,
pyridone H-3), 6.79 (dd, J = 2.4, 9.1 Hz, 1H, indole H-6), 6.93 (d, J = 2.4 Hz, 1H,
indole H-4), 7.27–7.39 (m, 5H, C6H5), 7.38 (d, J = 9.1 Hz, 1H, indole H-7), 7.59
(d, J = 7.3 Hz, 1H, pyridone H-6), 7.70 (t, J = 60 Hz, 1H, CHF2); 13C NMR (CDCl3) d
13.9, 30.3, 55.6, 66.9, 101.8, 105.6, 112.4, 114.3, 115.4, 122.1, 128.2, 128.3,
128.5, 130.0, 130.8, 131.0, 134.9, 135.6, 147.9, 156.7, 160.3, 165.4, 170.2.
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-5-carbonyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid benzyl ester (13b): Product 13b was obtained as a
27. Chavatte, P.; Yous, S.; Marot, C.; Baurin, N.; Lesieur, D. J. Med. Chem. 2001, 44,
3223.
28. Experimental procedures and spectral data for compounds 10, 12–14, 15, 17–
19. General. Melting points were determined on a Thomas–Hoover capillary
apparatus and are uncorrected. Unless otherwise noted, infrared (IR) spectra
were recorded as films on NaCl plates using a Nicolet 550 Series II Magna FT-IR
spectrometer. 1H NMR and 13C NMR spectra were measured on a Bruker AM-
300 spectrometer. Microanalyses (MicroAnalytical Service Laboratory,
Department of Chemistry, University of Alberta) were performed for C, H and
yellow oil in 31% yield; IR (film) 1736 (ester), 1697 (amide) cmÀ1 1H NMR
;
(CDCl3) d 2.40 (s, 3H, CH3), 3.67 (s, 2H, CH2CO2Bn), 3.72 (s, 3H, OCH3), 5.10 (s,
2H, CH2Ph), 6.54 (d, J = 9.7 Hz, 1H, pyridone H-3), 6.71 (dd, J = 2.4, 9.1 Hz, 1H,
indole H-6), 6.91 (d, J = 2.4 Hz, 1H, indole H-4), 7.04 (d, J = 9.1 Hz, 1H, indole H-
7), 7.22–7.35 (m, 5H, C6H5), 7.60 (dd, J = 2.4, 9.7 Hz, 1H, pyridone H-4), 7.64 (t,
J = 60 Hz, 1H, CHF2), 8.06 (d, J = 2.4 Hz, 1H, pyridone H-6); 13C NMR (CDCl3) d
13.0, 30.4, 55.6, 66.9, 101.6, 112.2, 112.9, 114.2, 115.8, 121.2, 128.1, 128.3,
128.5, 130.2, 130.7, 135.6, 135.7, 136.2, 140.4, 156.2, 160.1, 164.4, 170.5.
General procedure for the synthesis of [1-(N-difluoromethyl-1,2-dihydropyrid-2-
one-4- or 5-carbonyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acids (14a–b):
Palladium-on-charcoal (25 mg of 10% w/w) was added to a solution of a benzyl
ester 13a or 13b (0.27 g, 0.56 mmol) in EtOAc (22 mL) and MeOH (4 mL). The
resulting suspension was flushed with argon followed by three consecutive
flushes with H2 gas to remove any air or argon from the hydrogenation flask.
The pressure in the hydrogenation flask was maintained at 45 psi with H2 gas
using a Parr apparatus. After shaking for 24 h at 25 °C, the H2 gas was released
from the hydrogenation flask, and the reaction mixture was filtered through a
Celite pad to remove any Pd/C catalyst. The filtrate was concentrated in vacuo
to afford the crude product which was purified by silica gel column
chromatography using hexanes–ethyl acetate (1:3, v/v in 1% acetic acid) as
eluent to furnish the title compound 14a or 14b. Some physical and
spectroscopic data for 14a–b are listed below.
N
and were within 0.4% of theoretical values for all elements listed.
Compounds 10, 12a–b, 13a–b, 15, 17a–b and 18a–b showed a single spot on
Macherey-Nagel Polygram Sil G/UV254 silica gel plates (0.2 mm) using a low,
medium, and highly polar solvent system, and no residue remained after
combustion, indicating a purity of >95%. Silica gel column chromatography was
performed using Merck silica gel 60 ASTM (70–230 mesh). 2-Chloro-4-
chloromethylpyridine (16a) was synthesized in 43% overall yield by an
analogous reduction of 2-chloropyridine-4-carbonyl chloride (11a) with
NaBH4 in water32 followed by the reaction with SOCl2 in toluene.33 All other
reagents, purchased from the Aldrich Chemical Company (Milwaukee, WI),
were used without further purification. The in vivo anti-inflammatory assay
was carried out using protocols approved by the Health Sciences Animal
Welfare Committee at the University of Alberta.
(5-Methoxy-2-methyl-1H-indol-3-yl)acetic acid benzyl ester (10):18 Benzyl
chloroformate (0.65 mL, 4.6 mmol) was added to a solution of 5-methoxy-2-
methylindole-3-acetic acid (9, 1.0 g, 4.6 mmol) and triethylamine (0.7 mL,
5.0 mmol) in dichloromethane (15 mL) at 0 °C and the mixture was stirred for
5 min. DMAP (55 mg, 0.45 mmol) was added at 0 °C and the reaction was
allowed to proceed with stirring for 0.5 h. The reaction mixture was diluted
with dichloromethane (30 mL), washed successively with saturated NaHCO3
(30 mL), 0.1 N HCl (15 mL) and brine (30 mL), and the organic fraction was
dried (MgSO4). Filtration and then removal of the solvent from the organic
fraction in vacuo afforded the benzyl ester 10 as a yellow oil in 97% yield; 1H
NMR (CDCl3) d 2.37 (s, 3H, CH3), 3.71 (s, 2H, CH2CO2Bn), 3.78 (s, 3H, OCH3), 5.12
(s, 2H, CH2Ph), 6.77 (dd, J = 2.4, 8.5 Hz, 1H, H-6), 6.98 (d, J = 2.4 Hz, 1H, H-4),
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-4-carbonyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid (14a): Product 14a was obtained as a yellow solid in
75% yield; mp 177–179 °C; IR (film) 1728 (ester), 1653 (amide) cmÀ1 1H NMR
;
(DMSO-d6) d 2.29 (s, 3H, CH3), 3.67 (s, 2H, CH2CO2H), 3.78 (s, 3H, OCH3), 6.60
(dd, J = 1.8, 7.3 Hz, 1H, pyridone H-5), 6.79 (d, J = 1.8 Hz, 1H, pyridone H-3),
6.83 (dd, J = 2.4, 9.1 Hz, 1H, indole H-6), 7.06 (d, J = 2.4 Hz, 1H, indole H-4), 7.51
(d, J = 9.1 Hz, 1H, indole H-7), 7.89 (t, J = 60 Hz, 1H, CHF2), 8.01 (d, J = 7.3 Hz,

5780
M. A. Chowdhury et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5776–5780
1H, pyridone H-6), 12.42 (s, 1H, CO2H, exchangeable with D2O). Anal. Calcd for
19H16F2N2O5: C, 58.46; H, 4.13; N, 7.18. Found: C, 58.26; H, 4.20; N, 7.02.
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-5-carbonyl)-5-methoxy-2-methyl-
(s, 3H, OCH3), 5.03 (s, 2H, NCH2), 5.96 (d, J = 7.9 Hz, 1H, pyridone H-5), 5.97 (s,
C
1H, pyridone H-3), 6.80 (dd, J = 2.4, 8.5 Hz, 1H, indole H-6), 7.00 (d, J = 8.5 Hz,
1H, indole H-7), 7.04 (d, J = 2.4 Hz, 1H, indole H-4), 7.36 (d, J = 7.9 Hz, 1H,
pyridone H-6), 7.61 (t, J = 60 Hz, 1H, CHF2); 13C NMR (CDCl3) d 10.3, 30.5, 45.7,
51.9, 55.9, 100.9, 105.2, 105.4, 109.3, 111.4, 117.3, 128.4, 129.8, 131.2, 134.2,
152.6, 154.6, 160.7, 172.2.
1H-indol-3-yl]acetic acid (14b): Product 14b was obtained as a yellow solid in
46% yield; mp 163–165 °C; IR (film) 1725 (ester), 1676 (amide) cmÀ1 1H NMR
;
(DMSO-d6) d 2.33 (s, 3H, CH3), 3.66 (s, 2H, CH2CO2H), 3.76 (s, 3H, OCH3), 6.64 (d,
J = 9.7 Hz, 1H, pyridone H-3), 6.75 (dd, J = 2.4, 9.1 Hz, 1H, indole H-6), 7.04 (d,
J = 2.4 Hz, 1H, indole H-4), 7.23 (d, J = 9.1 Hz, 1H, indole H-7), 7.70 (dd, J = 2.4,
9.7 Hz, 1H, pyridyl H-4), 7.86 (t, J = 60 Hz, 1H, CHF2), 8.19 (d, J = 2.4 Hz, 1H,
pyridone H-6), 12.37 (s, 1H, CO2H, exchangeable with D2O); Anal. Calcd for
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-5-ylmethyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid methyl ester (18b): Product 18b was obtained as a
greenish oil in 53% yield; IR (film) 1734 (ester), 1690 (amide) cmÀ1 1H NMR
;
(CDCl3) d 2.36 (s, 3H, CH3), 3.67 (s, 3H, CO2CH3) 3.70 (s, 2H, CH2CO2CH3), 3.87
(s, 3H, OCH3), 5.01 (s, 2H, NCH2), 6.49 (d, J = 9.7 Hz, 1H, pyridone H-4), 6.83 (dd,
J = 2.4, 8.5 Hz, 1H, indole H-6), 7.01–7.11 (m, 4H, indole H-4, H-7, pyridone H-3,
H-6), 7.62 (t, J = 60 Hz, 1H, CHF2); 13C NMR (CDCl3) d 10.5, 30.5, 43.5, 51.9, 55.9,
100.8, 105.3, 109.3, 111.4, 117.2, 122.5, 125.9, 128.4, 131.2, 134.2, 140.0, 154.5,
160.4, 172.2.
C19H16F2N2O5: C, 58.46; H, 4.13; N, 7.18. Found: C, 58.29; H, 4.23; N, 7.23.
(5-Methoxy-2-methyl-1H-indol-3-yl)acetic acid methyl ester (15):22 Acetyl
chloride (0.23 mL, 3.12 mmol) was added to a solution of the acetic acid 9
(0.5 g, 2.28 mmol) in methanol (5 mL) at 0 °C, and the reaction was allowed to
proceed with stirring at 60 °C for 16 h. The mixture was then concentrated in
vacuo to dryness to which a solution of saturated aqueous NaHCO3 (20 mL)
was added. The solution was extracted with EtOAc (3 Â 20 mL), the combined
EtOAc extracts were successively washed with water and brine, and the
organic fraction was dried (MgSO4). Filtration and then removal of the solvent
in vacuo from the organic fraction afforded the methyl ester 15 as a pale yellow
oil in 95% yield; 1H NMR (CDCl3) d 2.39 (s, 3H, CH3), 3.67 (s, 2H, CH2CO2CH3),
3.68 (s, 3H, CO2CH3) 3.86 (s, 3H, OCH3), 6.78 (dd, J = 2.4, 8.5 Hz, 1H, H-6), 6.99
(d, J = 2.4 Hz, 1H, H-4), 7.16 (d, J = 8.5 Hz, 1H, H-7), 7.77 (br s, 1H, NH,
exchangeable with D2O).
General procedure for the synthesis of [1-(N-difluoromethyl-1,2-dihydropyrid-2-
one-4- or 5-ylmethyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acids (19a–b):
Aqueous LiOH (2 N, 5 mL) was added to a solution of a methyl ester 18a or 18b
(0.18 g, 0.46 mmol) in MeOH (5 mL) and THF (5 mL), and the reaction was
allowed to proceed at reflux for 3 h. The mixture was cooled to 25 °C, and
acidified to pH 3 using 5% HCl prior to extraction with EtOAc (3 Â 25 mL). The
combined organic extracts were successively washed with water (50 mL) and
brine, and the organic fraction was dried (MgSO4). Filtration and then removal
of the solvent from the organic fraction in vacuo afforded the crude product
which was purified by silica gel column chromatography using hexanes–ethyl
acetate (1:3, v/v in 1% acetic acid) as eluent to furnish the title compound 19a
or 19b. Some physical and spectroscopic data for 19a–b are listed below.
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-4-ylmethyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid (19a): Product 19a was obtained as a brown solid in
General procedure for the synthesis of [1-(2-chloropyridin-4- or 5-ylmethyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetic acid methyl esters (17a–b): A solution of
t-BuOK (254 mg, 2.15 mmol) in dry THF (3 mL) was added slowly via a syringe
to a stirred solution of 5-methoxy-2-methyl-1H-indol-3-yl)acetic acid methyl
ester (15, 0.5 g, 2.15 mmol) and either 2-chloro-4-chloromethylpyridine (16a)
or 2-chloro-5-chloromethylpyridine (16b) (0.35 g, 2.15 mmol) in dry THF
(30 mL) at 25 °C under argon. The reaction mixture was heated at reflux under
argon atmosphere for 60 h. The reaction mixture was cooled to 25 °C, poured
onto ice water, acidified with 5% aqueous HCl, and extracted with EtOAc
(3 Â 25 mL). The combined organic extracts were washed successively with
water and brine, and the organic fraction was dried (MgSO4). Filtration and
removal of the solvent from the organic fraction in vacuo afforded the impure
product which was purified by silica gel flash column chromatography using
hexanes–ethyl acetate (2:1, v/v) as eluent to furnish the respective title
compound 17a or 17b. Some physical and spectroscopic data for 17a–b are
listed below.
59% yield; mp 165–167 °C; IR (film) 1716 (ester), 1682 (amide) cmÀ1 1H NMR
;
(DMSO-d6) d 2.25 (s, 3H, CH3), 3.62 (s, 2H, CH2CO2H), 3.87 (s, 3H, OCH3), 5.28 (s,
2H, NCH2), 5.61 (d, J = 1.8 Hz, 1H, pyridone H-3), 6.09 (dd, J = 1.8, 7.9 Hz, 1H,
pyridone H-5), 6.70 (dd, J = 2.4, 9.1 Hz, 1H, indole H-6), 6.98 (d, J = 2.4 Hz, 1H,
indole H-4), 7.24 (d, J = 9.1 Hz, 1H, indole H-7), 7.75 (d, J = 7.9 Hz, 1H, pyridone
H-6), 7.76 (t, J = 60 Hz, 1H, CHF2), 12.10 (br s, 1H, CO2H, exchangeable with
D2O). Anal. Calcd for C19H18F2N2O4: C, 60.64; H, 4.82; N, 7.44. Found: C, 60.28;
H, 4.89; N, 7.38.
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-5-ylmethyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid (19b): Product 19b was obtained as a yellow solid in
85% yield; mp 145–147 °C; IR (film) 1714 (ester), 1688 (amide) cmÀ1 1H NMR
;
[1-(2-Chloropyridin-4-ylmethyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
methyl ester (17a): Product 17a was obtained as a brown oil in 34% yield; IR
(CDCl3) d 2.34 (s, 3H, CH3), 3.71 (s, 2H, CH2CO2H), 3.87 (s, 3H, OCH3), 5.00 (s, 2H,
NCH2), 6.48 (d, J = 9.1 Hz, 1H, pyridone H-4), 6.82 (dd, J = 2.4, 8.5 Hz, 1H, indole
H-6), 7.01–7.15 (m, 4H, indole H-4, H-7, pyridone H-3, H-6), 7.61 (t, J = 60 Hz,
1H, CHF2), 11.01 (br s, 1H, CO2H, exchangeable with D2O). Anal. Calcd for
(film) 1734 (ester) cmÀ1 1H NMR (CDCl3) d 2.31 (s, 3H, CH3), 3.69 (s, 3H,
;
CO2CH3) 3.73 (s, 2H, CH2CO2CH3), 3.87 (s, 3H, OCH3), 5.24 (s, 2H, NCH2), 6.75
(dd, J = 1.3, 5.5 Hz, 1H, pyridyl H-5), 6.80 (dd, J = 2.4, 8.5 Hz, 1H, indole H-6),
6.92 (d, J = 1.3 Hz, 1H, pyridyl H-3), 6.99 (d, J = 8.5 Hz, 1H, indole H-7), 7.06 (d,
J = 2.4 Hz, 1H, indole H-4), 8.27 (d, J = 5.5 Hz, 1H, pyridyl H-6); 13C NMR (CDCl3)
d 10.4, 30.5, 45.4, 52.0, 55.9, 100.8, 105.2, 109.3, 111.3, 119.7, 121.4, 128.4,
131.2, 134.3, 150.1, 150.5, 152.2, 154.6, 172.2.
C
19H18F2N2O4: C, 60.64; H, 4.82; N, 7.44. Found: C, 60.32; H, 4.84; N, 7.31.
29. 5-Lipoxygenase inhibition assay: The ability of the test compounds listed in
Table 1 to inhibit potato 5-LOX (Catalog No. 60401, Cayman Chemical, Ann
Arbor, MI, USA) (IC50 values, lM) were determined using an enzyme immuno
assay (EIA) kit (Catalog No. 760700, Cayman Chemical, Ann Arbor, MI, USA)
[1-(2-Chloropyridin-5-ylmethyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid
methyl ester (17b): Product 17b was obtained as a brown oil in 49% yield; IR
according to our previously reported method.34
30. Cyclooxygenase inhibition assays: The ability of the test compounds listed in
(film) 1735 (ester) cmÀ1
;
1H NMR (CDCl3) d 2.33 (s, 3H, CH3), 3.68 (s, 3H,
Table 1 to inhibit ovine COX-1 and human recombinant COX-2 (IC50 value, lM)
CO2CH3) 3.71 (s, 2H, CH2CO2CH3), 3.87 (s, 3H, OCH3), 5.27 (s, 2H, NCH2), 6.80
(dd, J = 2.4, 9.1 Hz, 1H, indole H-6), 7.01–7.11 (m, 3H, indole H-4, H-7, pyridyl
H-4), 7.19 (d, J = 7.9 Hz, 1H, pyridyl H-3), 8.20 (d, J = 1.8 Hz, 1H, pyridyl H-6);
13C NMR (CDCl3) d 10.4, 30.5, 43.8, 51.9, 55.9, 100.8, 105.1, 109.4, 111.3, 124.5,
128.4, 131.2, 132.4, 134.3, 136.6, 147.6, 150.6, 154.5, 172.2.
General procedure for the synthesis of [1-(N-difluoromethyl-1,2-dihydropyrid-2-
one-4- or 5-ylmethyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid methyl
esters (18a–b): Compounds 18a and 18b were synthesized using the same
procedure used for the synthesis of 13a–b. The product was purified by silica
gel flash chromatography using hexanes–ethyl acetate (1:2, v/v) as eluent to
furnish the respective title compound 18a or 18b. Some physical and
spectroscopic data for 18a–b are listed below.
were determined using an enzyme immuno assay (EIA) kit (Catalog No.
560131, Cayman Chemical, Ann Arbor, MI, USA) according to our previously
reported method.35
31. Anti-inflammatory assay: The test compounds 14a–b, 19a–b, and the reference
drug indomethacin were evaluated using the in vivo carrageenan-induced rat
foot paw edema model reported previously.36
32. Read, M. W.; Ray, P. S. J. Heterocycl. Chem. 1995, 32, 1595.
33. Zhuang, L.; Wai, J. S.; Embrey, M. W.; Fisher, T. E.; Egbertson, M. S.; Payne, L. S.;
Guare, J. P., Jr.; Vacca, J. P.; Hazuda, D. J.; Felock, P. J.; Wolfe, A. L.; Stillmock, K.
A.; Witmer, M. V.; Moyer, G.; Schleif, W. A.; Gabryelski, L. J.; Leonard, Y. M.;
Lynch, J. J., Jr.; Michelson, S. R.; Young, S. D. J. Med. Chem. 2003, 46, 453.
34. Chowdhury, M. A.; Abdellatif, K. R. A.; Dong, Y.; Das, D.; Suresh, M. R.; Knaus, E.
E. Bioorg. Med. Chem. Lett. 2008, 18, 6138.
[1-(N-Difluoromethyl-1,2-dihydropyrid-2-one-4-ylmethyl)-5-methoxy-2-methyl-
1H-indol-3-yl]acetic acid methyl ester (18a): Product 18a was obtained as a
35. Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem. 2003, 46,
4872.
brown oil in 34% yield; IR (film) 1734 (ester), 1684 (amide) cmÀ1 1H NMR
;
(CDCl3) d 2.32 (s, 3H, CH3), 3.69 (s, 3H, CO2CH3) 3.71 (s, 2H, CH2CO2CH3), 3.87
36. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.